We continue our studies on structure function relationship of human Interleukin-2. We have improved our techniques by developing PCR technique to do site-directed mutagenesis. We have made 11 mutants. Four of them are double substitutions or triple substitutions of cysteine residues to investigate the role of individual cys on IL-2 activity. We found that double substitution of cys at 58 and 125 positions (ala 58/125) has more activity than single substitution at 58 (ala58). This result suggested that the low activity of ala58 is probably due to the formation of wrong disulfide bond. In addition, we made several mutants by moving the cys to different positions to determine the loop size of the disulfide bond. This work is still underway.